3 Methodologies to Simulate Microgravity for Microbial Experiments

Introduction

Microgravity is a condition in which cells (and everything with mass!) experience gravitational forces lower than those on Earth, making them appear weightless or floating. This occurs when cells are in LEO, aboard the International Space Station (ISS) or during parabolic flights. Though gravity is still present, it is significantly reduced. For example, the gravitational force on Earth, the Moon and Mars is 9.81, 1.62 and 3.72 m/s2, respectively. In other words, if a cell on Earth weighs 100 ng, it would weigh 16.5 and 38 ng on the Moon and Mars, respectively.

Studying the effects of microgravity on cells is important because it allows biologists to study the effects of reduced gravitational forces on living organisms in specific areas such as:

• Cellular and Molecular Biology: Microgravity alters gene expression, protein production, and cell signaling pathways.
• Human Physiology: Studies on astronauts help understand muscle atrophy, bone density loss, and cardiovascular adaptations, which inform countermeasures for long-duration space missions and health applications on Earth.
• Microbial Behavior: Bacteria and fungi exhibit changes in growth rates, biofilm formation, and antibiotic resistance in microgravity, impacting spaceflight safety and biotechnology.
• Plant Growth and Agriculture: Understanding how plants adapt to microgravity aids in developing sustainable food production systems for deep-space exploration.
• Biopharmaceuticals: Microgravity enables the crystallization of proteins in ways not possible on Earth, improving drug design and medical research.

Microgravity analogs

Since conducting experiments in space is costly and logistically challenging, we use microgravity analogs on Earth to simulate weightless conditions and study bacterial responses. In other words, we simulate the effects of microgravity on cells, not microgravity itself. These models help investigate cell growth, gene expression, biofilm formation, and antibiotic resistance in bacteria under low-shear or altered gravity conditions.

• Rotating Wall Vessel (RWV) Bioreactors and Rotating Cell Culture Systems (RCCS)

Developed by NASA, Rotating Wall Vessels (RWVs), also called High Aspect Ratio Vessels (HARVs) or Slow Rotating Lateral Vessels (SRLVs), simulate microgravity by continuously rotating a culture around a horizontal axis, keeping bacteria in suspension, reducing sedimentation and shear stress, mimicking space-like conditions. They are widely used because the low-shear fluid conditions used resemble the spaceflight environment. Multiple different studies have shown that cells grown with this system exhibit enhanced biofilm formation, cell aggregation, and changes in antibiotic resistance and pathogenicity. REFS

• Clinostats

A clinostat rotates a sample along one or two axes to continuously change the direction of gravity’s pull, preventing the bacteria from sensing a fixed gravitational field. 2D clinostats rotate around a single axis, while 3D clinostats rotate in multiple directions, better mimicking microgravity. These devices are more affordable and simpler than RWV bioreactors, can be used for long-term experiments and in some cases allow researchers to run treatment and control conditions in parallel. They’re main application is to assess bacterial growth, motility, and gene expression.

• Random Positioning Machine (RPM)

The Random Positioning Machine (RPM) rotates a sample randomly along multiple axes, ensuring that the bacteria experience continuous changes in gravity vector direction. This instrument effectively reduces gravity to near 10⁻³ g, simulating long-term microgravity exposure. Compared to other analogs, it creates a more accurate simulation of microgravity conditions.

• Parabolic Flights

Aircraft (such as NASA’s “Vomit Comet” or ESA’s Zero-G Airbus) fly in parabolic arcs, creating brief periods (15-25 seconds) of microgravity during free fall. This is the closest Earth-based method to true microgravity in space and it allows direct comparison between normal gravity, hypergravity, and microgravity conditions.

• Magnetic Levitation

These analogs use superconducting magnets to create a force that counteracts Earth’s gravity, suspending bacterial cultures in a state that mimics weightlessness. One advantage over other analogs is that it provides weightlessness without rotation-induced shear stress. Although it is less commonly used for bacteria, it is being explored for cell biology studies.

Experimental Design

Define the research question or hypothesis.

Start with questions such as: How does microgravity affect bacterial growth rate and cell size? Do pathogenic bacteria become more virulent in microgravity? Does microgravity enhance bacterial biofilm formation? Are bacteria more resistant to antibiotics in space? Once you select your question, formulate a hypothesis: “In simulated microgravity, Pseudomonas aeruginosa will form denser biofilms compared to normal gravity conditions.”

Selection of the model and culture media

Once you have a hypothesis, identify which specie is more appropriate to test it. Start with those species with “space legacy”, this is, that have been previously used in real or simulated microgravity experiments. Consider other factors such as: Pathogenic bacteria (e.g. Salmonella typhimurium, Escherichia coli, Staphylococcus aureus), biofilm-forming bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae), spore-forming species (Bacillus subtilis), extremophiles (Deinococcus radiodurans), among others. Next identify the optimal growth conditions, starting with the growth media: Nutrient Broth/Agar for general-purpose bacterial growth, Luria-Bertani (LB) for enteric bacteria such as E. coli, urea-Based medium for Sporosarcina pasteurii, tryptic Soy Broth (TSB) + Glucose for biofilm studies, among others.

Selection of the microgravity analog

When choosing the analog for your experiments, consider the following advantages and disadvantages:

Experiment setup and sample preparation

Step 1: Culture Preparation

Inoculate bacterial cultures into sterile growth media and grow for ~ 15 h at its optimal temperature (30-37° C) under constant agitation. The day of the experiment, adjust the initial OD₆₀₀ (optical density at 600 nm) to ensure similar starting cell densities (~0.1–0.2 OD₆₀₀). Prepare the staring cell dilution (commonly 1:100) using the selected growth media. Prepare triplicates for each condition.

Step 2: Treatment (microgravity) and control (gravity) conditions

Transfer the bacteria suspensions to the appropriate growth vessel, depending on the analog used (HARVS for RWV or 2 mL screw-cap tubes for clinostats and RPM). Load the microgravity samples onto the device. For the control group, incubate the samples either under static conditions or onto the clinostat gravity section, if available.

Step 3: Incubation

Incubate both treatment and control under identical temperature, oxygen, and media conditions. Monitor growth for 24–48 hours or longer, depending on bacterial doubling time and the type of information you need to test your hypotheses.

Biological endpoints

The potential effects of simulated microgravity on bacteria cultures could be measured using different biological endpoints, based on the hypotheses I question:

• Growth Kinetics and viability

The first and most straightforward parameter for estimating bacterial growth is optical density at 600 nm (OD₆₀₀), which tracks turbidity over time. Measured using a spectrophotometer, OD₆₀₀ provides a rapid assessment of growth rate changes, such as faster or slower doubling times. However, it cannot differentiate between viable and dead cells. This measurement can be taken at predefined incubation times to represent specific growth phases (exponential, stationary, or death) or at regular intervals (minutes or hours) to construct a growth curve, revealing adjustments in lag phase duration or generation time during exponential growth.

To assess viability, colony-forming units per milliliter (CFU/mL) must be determined, quantifying only viable cells capable of forming visible colonies. This requires performing serial dilutions, plating on appropriate agar, and counting colonies. An increase in CFU may indicate enhanced survival or adaptation, while a decrease could suggest environmental stress.

• Changes in phenotype

Gravity influences bacterial swimming patterns and flagellar function, hence changes in motility and flagellar structure could be tracked using video microscopy or soft agar motility assays to determine any shift in motility or directional changes due to altered fluid shear forces. Another phenotype aspect commonly altered by microgravity is the formation and structure of biofilms, altered by changes in cell physiology leading to a higher production of extracellular polymeric substances (EPS) and to the alteration of fluid shear. Biofilm biomass can be measured using the crystal violet assay, while structure is generally studied with confocal laser scanning microscopy (CLSM).

• Antibiotic susceptibility & stress responses

The Minimum Inhibitory Concentration (MIC) testing is essential to evaluate how microgravity alters antibiotic resistance, and it is generally done using broth dilution or disc diffusion assays with various antibiotics. The expected outcome is an increase in resistance, potentially due to cell wall modifications or efflux pump activation. Additionally, stress-response gene expression can be analyzed through qPCR or RNA-Seq, as bacteria activate stress pathways under microgravity. This experiment requires the extraction of total RNA to quantify the expression of genes related to oxidative stress (sodA, katG), DNA repair (recA, lexA), and antibiotic resistance (efflux pump genes such as acrB, mexAB).

• Transcriptomic and proteomic analyses

Differential gene expression analysis using RNA-Seq helps identify genome-wide metabolic shifts under microgravity by comparing the gene expression profiles between bacteria grown under microgravity and gravity conditions. This approach directly quantifies the number of gene copies  coding for proteins related to cell division, metabolism, and virulence, shedding light on microbial adaptation mechanisms. Similarly, studying protein expression and post-translational modifications through mass spectrometry-based proteomics provides further insights into the synthesis of proteins involved in stress-related and biofilm-associated processes, highlighting potential survival strategies in altered gravitational environments.